The Respiratory Syncytial Virus (RSV) poses a serious threat to newborn children, the elderly and immuno-compromised patients; an effective vaccine has the potential to protect all vulnerable populations, but such a protective vaccine has not yet been developed. The RSV fusion (F) protein induces potent, neutralizing antibodies (NAbs) that protect from infection; the most potent of these NAbs bind only to its pre-fusion conformation (preF). preF as a soluble immunogen elicits the most potently protective responses, but use of recombinant, soluble preF as an immunogen has been hindered by its instability. Several groups have identified variants that stabilize F in its prefusion conformation, but none have achieved a satisfactory degree of stability. Avatar is developing a preF immunogen that is considerably more stable and that will provide potent, long-lasting, and cost-effective protection against RSV infection Avatar has developed a technology that introduces targeted, zero-length dityrosine (DT) crosslinks into fully folded, native proteins, preserving their structural integrity. DT locking involves 2 steps: (i) expressing and purifying soluble F proteins with targeted, conservative to-Tyr substitutions, and (ii) enzymatically crosslinking the complex in its pre-fusion conformation. DT bonds are used to lock together interactions between secondary and tertiary protein structures within the preF trimer that are beyond the reach of traditional protein engineering and can hence solve otherwise intractable protein engineering problems. Avatar's stabilized DT-preF immunogen will thus provide the required stability for storage and distribution, and thereby exceed the stability needed for successful product development; furthermore, DT-preF will focus immune responses on potently neutralizing epitopes that are only displayed in the preF conformation. As a result, vaccination with DT-preF will elicit potent and lasting protection against RSV. Our goal is to develop an immunogen with very high stability (half-life > 1yr) that elicits potent and protective Ab responses. To accomplish this goal, we will carry out the following Phase II Specific Aims: I. Make and test additional DT-preF designs. Introduce an additional crosslink at the base of the preF trimer. (Milestone: novel DT-preF molecules antigenically intact, and have improved stability.) II. Proteolytically remove the trimerization motif via engineered cleavage sites. (Milestone: DT-preF molecules are antigenically intact and stable; lead and back-up molecules are selected.) III. Confirm prefusion conformation of the DTpreF immunogen. MS and crystallographic analysis (Milestones: Mass. spec of the lead and back-up DT-preF protein fragments shows DT bonds are formed at targeted positions, and crystallographic analysis confirms DT-preF proteins retain the preF conformation.) IV. Demonstrate potency in animal models. Immunogencity/histopathology study in mice and cotton rats. (Milestones: lead and back-up DT-preF generate high neutralizing titers and protection from challenge.) In Phase III we will manufacture DT-preF under cGMP and perform pre-clinical testing safety, with a view toward filing an IND with the FDA.